scholarly journals 151 Potentiating the Large-Scale Expansion and Engineering of Peripheral Blood-Derived CAR NK Cells for Off-the-Shelf Application

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A159-A159
Author(s):  
Michael Whang ◽  
Ming-Hong Xie ◽  
Kate Jamboretz ◽  
Hadia Lemar ◽  
Chao Guo ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Therapy ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Adoptive Cell Therapy ◽  
Therapeutic Window ◽  
Target Cells ◽  
Healthy Donors ◽  
Custom Made

BackgroundPeripheral blood natural killer (NK) cells are mature cytotoxic innate lymphocytes possessing an inherent capacity for tumor cell killing, thus making them attractive candidates for adoptive cell therapy. These NK cells are also amenable to CRISPR and chimeric antigen receptor (CAR) genomic engineering for enhanced functions. Moreover, NK cells possess an inherent capacity for off-the-shelf therapy since they are not known to cause graft-versus-host disease, unlike T cells. Presently, approved CAR cell therapy is custom-made from each patient‘s own T cells, a process that can limit patient pool, narrow therapeutic window, and contribute to product variability. In this study, we investigate whether peripheral blood NK cells from a selected donor can be edited, engineered, and expanded sufficiently for off-the-shelf use in a wide patient population.MethodsUsing the CRISPR/Cas9 system, we knocked out CISH expression in isolated peripheral blood NK cells from 3 healthy donors. Subsequently, we expanded edited NK cells by using IL-2 and sequential stimulations using NKSTIM, a modified K562 stimulatory cell line expressing membrane-bound form of IL-15 (mbIL-15) and 4-1BBL. IL-12 and IL-18 were added twice during expansion to drive memory-like NK cell differentiation. We transduced the expanded NK cells to express engineered CD19-targeted CAR and mbIL-15 during an interval between the first and second NKSTIM pulses. We assessed NK cell cytotoxicity against Nalm6 target cells by IncuCyte.ResultsIsolated peripheral blood NK cells from 3 healthy donors were successfully edited using CRISPR/Cas9, engineered to express high levels of CAR, extensively expanded using a series of NKSTIM pulses in the presence of IL-2, and differentiated into memory-like NK cells using IL-12 and IL-18. Interestingly, NK cells from the 3 donors exhibited distinct outcomes. NK cells from one donor reached a peak expansion limit of approximately 7-million-fold before undergoing contraction whereas NK cells from two donors continued to expand over the length of the study surpassing 100-million-fold expansion, without appearing to have reached a terminal expansion limit. At the end of the study, NK cells from one donor exceeded 1-billion-fold expansion and maintained 88% cytolytic activity compared to Nkarta’s standard process control in a 72-hour IncuCyte assay.ConclusionsIn this study, we demonstrate that healthy donor-derived peripheral blood NK cells are capable of expanding over billion-fold while maintaining potency. These results provide a rationale for the development of off-the-shelf CAR NK cell therapies using NK cells from donors selected to provide optimal product characteristics.Ethics ApprovalHuman samples were collected with written informed consent by an approved vendor.

scholarly journals Genetically Modified T Cells for Esophageal Cancer Therapy: A Promising Clinical Application

2021 ◽  
Vol 11 ◽  
Author(s):  
Yu-Ge Zhu ◽  
Bu-Fan Xiao ◽  
Jing-Tao Zhang ◽  
Xin-Run Cui ◽  
Zhe-Ming Lu ◽  
...  
Keyword(s):  
T Cells ◽  
Esophageal Cancer ◽  
T Cell ◽  
Cancer Therapy ◽  
Cell Therapy ◽  
Nk Cells ◽  
Nk Cell ◽  
Genetically Modified ◽  
Adoptive Cell Therapy ◽  
T Cell Therapy

Esophageal cancer is an exceedingly aggressive and malignant cancer that imposes a substantial burden on patients and their families. It is usually treated with surgery, chemotherapy, radiotherapy, and molecular-targeted therapy. Immunotherapy is a novel treatment modality for esophageal cancer wherein genetically engineered adoptive cell therapy is utilized, which modifies immune cells to attack cancer cells. Using chimeric antigen receptor (CAR) or T cell receptor (TCR) modified T cells yielded demonstrably encouraging efficacy in patients. CAR-T cell therapy has shown robust clinical results for malignant hematological diseases, particularly in B cell-derived malignancies. Natural killer (NK) cells could serve as another reliable and safe CAR engineering platform, and CAR-NK cell therapy could be a more generalized approach for cancer immunotherapy because NK cells are histocompatibility-independent. TCR-T cells can detect a broad range of targeted antigens within subcellular compartments and hold great potential for use in cancer therapy. Numerous studies have been conducted to evaluate the efficacy and feasibility of CAR and TCR based adoptive cell therapies (ACT). A comprehensive understanding of genetically-modified T cell technologies can facilitate the clinical translation of these adoptive cell-based immunotherapies. Here, we systematically review the state-of-the-art knowledge on genetically-modified T-cell therapy and provide a summary of preclinical and clinical trials of CAR and TCR-transgenic ACT.

scholarly journals Recognition of virus-infected cells by natural killer cell clones is controlled by polymorphic target cell elements.

1993 ◽  
Vol 178 (3) ◽  
pp. 961-969 ◽  
Author(s):  
M S Malnati ◽  
P Lusso ◽  
E Ciccone ◽  
A Moretta ◽  
L Moretta ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Target Cell ◽  
Nk Cell ◽  
Class I ◽  
Target Cells ◽  
Cell Recognition ◽  
Infected Cells ◽  
Nk Cell Recognition

Natural killer (NK) cells provide a first line of defense against viral infections. The mechanisms by which NK cells recognize and eliminate infected cells are still largely unknown. To test whether target cell elements contribute to NK cell recognition of virus-infected cells, human NK cells were cloned from two unrelated donors and assayed for their ability to kill normal autologous or allogeneic cells before and after infection by human herpesvirus 6 (HHV-6), a T-lymphotropic herpesvirus. Of 132 NK clones isolated from donor 1, all displayed strong cytolytic activity against the NK-sensitive cell line K562, none killed uninfected autologous T cells, and 65 (49%) killed autologous T cells infected with HHV-6. A panel of representative NK clones from donors 1 and 2 was tested on targets obtained from four donors. A wide heterogeneity was observed in the specificity of lysis of infected target cells among the NK clones. Some clones killed none, some killed only one, and others killed more than one of the different HHV-6-infected target cells. Killing of infected targets was not due to complete absence of class I molecules because class I surface levels were only partially affected by HHV-6 infection. Thus, target cell recognition is not controlled by the effector NK cell alone, but also by polymorphic elements on the target cell that restrict NK cell recognition. Furthermore, NK clones from different donors display a variable range of specificities in their recognition of infected target cells.

scholarly journals Slaying the Trojan Horse: Natural Killer Cells Exhibit Robust Anti-HIV-1 Antibody-Dependent Activation and Cytolysis against Allogeneic T Cells

2014 ◽  
Vol 89 (1) ◽  
pp. 97-109 ◽  
Author(s):  
Shayarana L. Gooneratne ◽  
Jonathan Richard ◽  
Wen Shi Lee ◽  
Andrés Finzi ◽  
Stephen J. Kent ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Target Cells ◽  
Dependent Manner ◽  
Inhibitory Receptors ◽  
Cell Responses ◽  
Anti Hiv ◽  
Hiv 1

ABSTRACTMany attempts to design prophylactic human immunodeficiency virus type 1 (HIV-1) vaccines have focused on the induction of neutralizing antibodies (Abs) that block infection by free virions. Despite the focus on viral particles, virus-infected cells, which can be found within mucosal secretions, are more infectious than free virus bothin vitroandin vivo. Furthermore, assessment of human transmission couples suggests infected seminal lymphocytes might be responsible for a proportion of HIV-1 transmissions. Although vaccines that induce neutralizing Abs are sought, only some broadly neutralizing Abs efficiently block cell-to-cell transmission of HIV-1. As HIV-1 vaccines need to elicit immune responses capable of controlling both free and cell-associated virus, we evaluated the potential of natural killer (NK) cells to respond in an Ab-dependent manner to allogeneic T cells bearing HIV-1 antigens. This study presents data measuring Ab-dependent anti-HIV-1 NK cell responses to primary and transformed allogeneic T-cell targets. We found that NK cells are robustly activated in an anti-HIV-1 Ab-dependent manner against allogeneic targets and that tested target cells are subject to Ab-dependent cytolysis. Furthermore, the educated KIR3DL1+NK cell subset from HLA-Bw4+individuals exhibits an activation advantage over the KIR3DL1−subset that contains both NK cells educated through other receptor/ligand combinations and uneducated NK cells. These results are intriguing and important for understanding the regulation of Ab-dependent NK cell responses and are potentially valuable for designing Ab-dependent therapies and/or vaccines.IMPORTANCENK cell-mediated anti-HIV-1 antibody-dependent functions have been associated with protection from infection and disease progression; however, their role in protecting from infection with allogeneic cells infected with HIV-1 is unknown. We found that HIV-1-specific ADCC antibodies bound to allogeneic cells infected with HIV-1 or coated with HIV-1 gp120 were capable of activating NK cells and/or trigging cytolysis of the allogeneic target cells. This suggests ADCC may be able to assist in preventing infection with cell-associated HIV-1. In order to fully utilize NK cell-mediated Ab-dependent effector functions, it might also be important that educated NK cells, which hold the highest activation potential, can become activated against targets bearing HIV-1 antigens and expressing the ligands for self-inhibitory receptors. Here, we show that with Ab-dependent stimulation, NK cells expressing inhibitory receptors can mediate robust activation against targets expressing the ligands for those receptors.

scholarly journals Natural Born Killers: NK Cells in Cancer Therapy

Cancers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 2131 ◽  
Author(s):  
S. Elizabeth Franks ◽  
Benjamin Wolfson ◽  
James W. Hodge
Keyword(s):  
Cell Therapy ◽  
Nk Cells ◽  
Adoptive Transfer ◽  
Cellular Therapy ◽  
Nk Cell ◽  
Hematologic Malignancy ◽  
Adoptive Cell Therapy ◽  
Genetically Engineered ◽  
Surface Proteins ◽  
Independent Manner

Cellular therapy has emerged as an attractive option for the treatment of cancer, and adoptive transfer of chimeric antigen receptor (CAR) expressing T cells has gained FDA approval in hematologic malignancy. However, limited efficacy was observed using CAR-T therapy in solid tumors. Natural killer (NK) cells are crucial for tumor surveillance and exhibit potent killing capacity of aberrant cells in an antigen-independent manner. Adoptive transfer of unmodified allogeneic or autologous NK cells has shown limited clinical benefit due to factors including low cell number, low cytotoxicity and failure to migrate to tumor sites. To address these problems, immortalized and autologous NK cells have been genetically engineered to express high affinity receptors (CD16), CARs directed against surface proteins (PD-L1, CD19, Her2, etc.) and endogenous cytokines (IL-2 and IL-15) that are crucial for NK cell survival and cytotoxicity, with positive outcomes reported by several groups both preclinically and clinically. With a multitude of NK cell-based therapies currently in clinic trials, it is likely they will play a crucial role in next-generation cell therapy-based treatment. In this review, we will highlight the recent advances and limitations of allogeneic, autologous and genetically enhanced NK cells used in adoptive cell therapy.

NK-Cell Reconstitution after HLA-Identical Blood and Marrow Transplantations for CML: The Recovery of NKG2D Is Inversely Correlated with NKG2A and It Promotes the GVL Effect.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4879-4879
Author(s):  
Juan Tong ◽  
Huilan Liu ◽  
Liangquan Geng ◽  
Zimin Sun ◽  
Baolin Tang ◽  
...  
Keyword(s):  
Nk Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Linear Regression Analysis ◽  
Cell Activity ◽  
Target Cells ◽  
Peripheral Blood Stem Cells ◽  
Peripheral Blood Mononuclear ◽  
Blood Stem Cells

Abstract Natural killer (NK) cell alloreactivity is reported to mediate strong graft versus leukemia (GVL) effect in patients after allogeneic stem-cell transplantation. NKG2D receptors recognize human MHC class Ichain related A and B (MICA/B) and UL16-binding protein 1∼4(ULBP 1∼4) on target cells, thereby regulating NK cell activity. To examine the recovery of NKG2D, NKG2A and other receptors expression by NK cells, we used flow cytometry to evaluate samples from 11 chronic myeloid leukemia patients and their donors in the year following unmanipulated HLA completely matched peripheral blood stem cells plus bone marrow transplantation. Peripheral blood mononuclear cells from patients and their donors were tested in standard 51Cr release assays against cultured K562 targets to determine the cytotoxicity of the NK cells in the same intervals. There is no mismatched immunoglobulin-like receptor (KIR) ligand in both GVH and HVG direction. The reconstitution of KIR2DL1 (CD158a) after this transplantation protocol was very slow and these receptors didn’t reach normal value in the year and KIR2DL2 (CD158b) was much better. The NKG2D increased and the NKG2A decreased quickly at the same time after engraftment, and used linear regression analysis we demonstrated that NKG2A recovery was inversely correlated with NKG2D recovery in the year following transplantation. The ratio of NKG2D/NKG2A was directly associated with the capacity of NK-cell cytotoxicity. Thus, the reconstitution of NKG2D makes contribution to the recovery of the NK cytotoxicity. These results reveals that the NK cells generated after HLA matched blood plus bone morrow transplantation of CML patients are promoted at an immature state characterized by specific phenotypic features and enhanced functioning, having potential impact for immune responsiveness and transplantation outcome.

Ex Vivo Activated Natural Killer (NK) Cells from Myeloma Patients Kill Autologous Myeloma and Killing Is Enhanced by Elotuzumab

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3666-3666
Author(s):  
Tarun K. Garg ◽  
Susann Szmania ◽  
Jumei Shi ◽  
Katie Stone ◽  
Amberly Moreno-Bost ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Therapy ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
K562 Cells ◽  
Scid Mice ◽  
Target Cells ◽  
Recent Trial ◽  
Nk Cell Therapy

Abstract Immune-based therapies may improve outcome for multiple myeloma (MM) by eradicating chemo-resistant disease. Our recent trial utilizing IL2 activated, killer immunoglobulin-like receptor-ligand mismatched NK cell transfusions from haplo-identical donors yielded (n) CR in 50% of patients. Unfortunately, after NK cell therapy, 2/10 patients had progressive disease, and the median duration of response for the other 8/10 patients was only 105 days (range 58–593). This may have been due to an insufficient dose of alloreactive NK cells and early rejection. Furthermore, appropriate donors were identified for only 30% of otherwise eligible patients. We therefore investigated whether NK cells from MM patients could be expanded and activated to kill autologous MM. We then examined whether pre-treatment of MM cell targets with elotuzumab, a humanized antibody to the MM tumor antigen CS1, could further enhance NK cell-mediated lysis. PBMC from 5 MM patients were co-cultured for 14 days with irradiated K562 cells transfected with 4-1BBL and membrane bound IL15 in the presence of IL2 (300U/ml) as previously described (Imai et al, Blood2005;106:376–383). The degree of NK cell expansion, NK immunophenotype, and ability to kill MM (4 hour 51Cr release assays) were assessed. To determine the ability of ex vivo expanded NK cells to traffic to bone marrow, activated NK cells were injected into the tail vein of NK cell depleted NOD-SCID mice, which were then sacrificed after 48 hours. Flow cytometry for human CD45, CD3, and CD56 was performed on cells from blood, marrow and spleen. There was an average 64-fold expansion of NK cells (range: 8–200) after 2 weeks of co-culture with K562 transfectants. Expansion of T cells was not observed. The NK cell activating receptor NKG2D, and natural cytotoxicity receptors NKp30, NKp44, and NKp46 were up-regulated following the expansion. Expanded NK cells were able to kill autologous MM (E:T ratio 10:1, average 31%, range 22–41%), whereas resting NK cells did not. Pretreatment of autologous MM cells with elotuzumab increased the activated NK cell-mediated killing by 1.7-fold over target cells pretreated with an isotype control antibody. This level of killing was similar to that of the highly NK kill-sensitive cell line K562 (Figure). Autologous PHA blasts and CD34+ stem cells were not killed. Activated human NK cells were detectable in the bone marrow of NOD-SCID mice 48 hours after injection. Ex vivo activation of NK cells from MM patients with K562 transfectants can induce killing of autologous MM and produce large numbers of NK cells for potential therapy. The addition of elotuzumab to activated NK cell therapy enhances anti-MM effects by ADCC thus invoking an additional NK cell-mediated mechanism of MM killing. Importantly, ex vivo activated NK cells traffic to the bone marrow in mice. Autologous NK cell therapy eliminates the issues related to allo-donor availability and early NK cell rejection, and could provide an option for patients refractory to chemotherapy agents. Figure Figure

In Vitro Generation of Cytotoxic T Lymphocytes against MAGE A1 and MAGE A3 Derived From Healthy Donors.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4079-4079
Author(s):  
Lei Bao ◽  
Mindy M Stamer ◽  
Kimberly Dunham ◽  
Deepa Kolaseri Krishnadas ◽  
Kenneth G Lucas
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Lymphocytes ◽  
Cell Therapy ◽  
Cytotoxic T Lymphocytes ◽  
Cd8 T Cells ◽  
Target Cells ◽  
Ifn Gamma ◽  
Healthy Donors

Abstract Abstract 4079 Poster Board III-1014 MAGE A1 and MAGE A3 are cancer testis antigens that are expressed on a number of malignant tumor cells, but not by normal cells, except for male germ cells which lack HLA expression. Therefore, MAGE cytotoxic T lymphocytes are strictly tumor-specific. Adoptive transfer of antigen specific cytotoxic T lymphocytes (CTL) provides immediate graft-versus tumor effects while minimizing risk for graft-versus-host disease. The aim of the current study was to find ideal conditions for expansion of CTL targeting tumor-associated antigens from peripheral blood mononuclear cells (PBMCs) of healthy donors to be used in allogenic cell therapy. In this study we investigated the ability to generate MAGE A1 and MAGE A3 specific cytotoxic T cells using autologous dendritic cells (DC) loaded with MAGE A1 and MAGE A3 overlapping peptides. CTL lines specific for MAGE A1 and MAGE A3 were established by stimulating CD8 T cells from healthy donors with autologous dendritic cells loaded with MAGE A1 or MAGE A3 overlapping pooled peptides in round-bottomed, 96-well plates. CD8+ T cells were restimulated with the same ratio of peptide pulsed DC on days 7 and 14 in the presence of IL-2 (50 U/ml), IL-7 and IL-15 (5 ng/ml). These microcultures were screened 10 days after the third stimulation for their capacity to produce interferon-gamma (IFN-gamma) when stimulated with autologous EBV-transformed B lymphocytes (BLCL) transduced with lentivirus(LV) encoding MAGE A1 or MAGE A3 and autologous BLCL transduced with LV encoding GFP. MAGE A1 and MAGE-A3 specific IFN-gamma producing cells were rapidly expanded in OKT3 and IL2. The specificity of the rapidly expanded MAGE A1 and MAGE A3 specific T cells was confirmed by IFN-gamma production as measured by intracellular cytokine staining and ELISA as well as antigen specific cytotoxicity by a standard 51chromium (51Cr) release assay. We successfully generated MAGE A1 and MAGE A3 specific CTL lines from healthy donors using this method. Specific CTL lines showed cytotoxicity in vitro not only to target cells pulsed with MAGE A1 or MAGE A3 peptides but also to target cells transduced with LV-MAGE A1 or LV-MAGE A3. Specific cytolytic activity was accompanied by IFN-gamma secretion. These data indicate that tumor antigen specific CTL can be expanded using overlapping peptides regardless of an individual's HLA specificity. The ability to generate tumor specific CTL from donors of various HLA backgrounds provide a rationale for utilizing MAGE A1 and MAGE A3 overlapping peptides for expansion of antigen specific T cells for adoptive T-cell therapy against MAGE A1 or MAGE A3 expressing tumors. Disclosures: No relevant conflicts of interest to declare.

Immune Function in Follicular Lymphoma (FL): Comparison of Patient with Her Healthy Identical Twin Reveals Enhanced NK Cell Responses in the Patient During Spontaneous Remission [KC1].

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 5012-5012
Author(s):  
Elena Gitelson ◽  
Alexander W. MacFarlane ◽  
Kerry S Campbell ◽  
R. Katherine Alpaugh ◽  
Tahseen I. Al-Saleem ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Line ◽  
Peripheral Blood ◽  
Partial Remission ◽  
Nk Cell ◽  
Identical Twin ◽  
Effector Memory ◽  
Target Cells ◽  
Tumor Target ◽  
Memory Phenotype

Abstract Abstract 5012 Identical twins are an excellent model in which to study tumor-specific immune responses (Gitelson et al Br J Haem 2002) as it can be postulated that the immune systems are identical. Spontaneous remissions in FL occur but the immunologic mechanisms remain elusive. We investigated adaptive and innate immunologic phenotypes and responses in a 41-year old patient with untreated grade 1 FL, and her healthy identical twin sister. Patients and methods The patient has had a waxing and waning course of FL over 7 years and currently is in spontaneous partial remission (> 50% reduction of generalized lymphadenopathy) and her peripheral blood was negative for t(14:18) by PCR at the time of this analysis. Immunologic responses of her twin, as well as another set of healthy identical twin sisters, were investigated as controls. We studied peripheral blood using 8-color multiparametric flow cytometry for frequencies and phenotypes of NK and regulatory T cells (Tregs). FOXP3+ cells were further analyzed for naïve, central memory and effector memory phenotype. Activation of NK cells and degranulation in response to tumor target cells as measured by Lysosomal-Associated Membrane Protein 1 (LAMP-1) surface expression were investigated. An erythroblastoid cell line (K562) and an EBV transformed MHC-I deficient lymphoblastoid cell line (721.221) were used as targets. Results NK studies revealed that activated CD69+ NK cells were increased in percentage in FL patient (4.43%) compared to her healthy twin (1.82%), and had increased mean fluorescence intensity (MFI) of 1057 vs 357, respectively. In contrast, almost identical frequencies and MFI of activated NK cells were found in the set of healthy twins (0.55% vs 0.54% and 153 vs 153, respectively). NK cells of FL patient exhibited elevated degranulation compared to the healthy twin in response to stimulation by either target cell: LAMP-1 staining MFI 1907 vs 1395, respectively for 721.221 target cells and 1394 vs 1122, respectively for K562 cells, whereas no difference was detected in non-stimulated NK cells (MFI 536 vs 506, respectively). In addition, Killer Cell Immunoglobulin-like Receptor (KIR) analysis revealed a deficit in the percentage of NK cells staining with an antibody recognizing KIR2DL1/S1 in the patient, but not in her healthy twin (7.0% vs 15.1% of NK cells, respectively), whereas no other differences in KIR receptor expression profiles were found for other KIR or in comparing the set of healthy twins. Analysis of frequencies of circulating Tregs revealed no difference between FL patient and her healthy twin: CD4+CD25+FOXP3+ cells represented 4.92% vs 5.78%, respectively of total CD4 cells and CD8+CD25+FOXP3+ cells represented 1.25% vs 1.37%, respectively of total CD8 cells. Analysis of T cell subsets revealed that Tregs in FL patient and in her twin were mainly of effector memory phenotype: CCR7-/CD45RA-/CD45RO+ (82.6% vs 74.4%, respectively) and central memory phenotype: CCR7+/CD45RA-/CD45RO+ (10.7 vs 14.3%, respectively). Conclusions NK cell studies of FL patient revealed increased numbers of activated CD69+ NK cells which correlated with increased degranulation response to tumor target cells and a deficit in the NK cell repertoire, as noted by the deficiency in NK cells expressing KIR2DL1/S1 in the patient compared to the healthy twin, while no differences in frequencies of circulating Tregs or Treg phenotypes were identified. Although the current results are based upon a single sampling, due to limited availability, the observed potentiation of NK cell activity in the patient with FL suggest a potentially important role of NK cells in tumor control during spontaneous partial remission. Follow-up studies of persisting remission, temporary flare-up or at disease progression will be of interest. Disclosures No relevant conflicts of interest to declare.

CSL362: A Monoclonal Antibody to Human Interleukin-3 Receptor (CD123), Optimized for NK Cell-Mediated Cytotoxicity of AML Stem Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3598-3598 ◽  
Author(s):  
Samantha J. Busfield ◽  
Mark Biondo ◽  
Mae Wong ◽  
Hayley S. Ramshaw ◽  
Erwin M Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Monoclonal Antibody ◽  
Mouse Model ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Research Funding ◽  
Nk Cell ◽  
Target Cells ◽  
Cell Leukemia ◽  
Interleukin 3

Abstract Abstract 3598 The interleukin-3 receptor alpha chain (IL-3Rα/CD123) is expressed in a variety of hematological malignancies including AML, MDS, B-ALL, Hodgkin's lymphoma, hairy cell leukemia, systemic mastocytosis, plasmacytoid dendritic cell leukemia and CML. In AML, the majority of AML blasts express CD123 and this receptor is selectively over expressed on CD34+CD38− leukemic stem cells (LSC) compared to normal hematopoietic stem cells. This difference may provide a biological advantage to the leukemic cells given the survival and proliferation-promoting activities of IL-3, whilst at the same time providing an opportunity to target these malignant cells selectively. We have shown previously that 7G3, a mouse monoclonal antibody (mAb) which blocks IL-3 binding to CD123, is capable of eliminating human LSC in a mouse model of human AML by a combination of mechanisms, including engagement of the innate immune system via Fc-dependent mechanisms (Jin et al., 2009 Cell Stem Cell, 5:31). We have subsequently humanised and affinity-matured this antibody and, in addition, have engineered the Fc-domain to optimise potential cytotoxicity against AML cells. The resultant antibody, CSL362, retains the ability to neutralise IL-3 and has enhanced affinity for the FcγRIIIa (CD16) on NK cells. In vitro studies have demonstrated that the increased affinity for CD16 correlates with greater antibody-dependent cell-mediated cytotoxicity (ADCC) against CD123 expressing cell lines compared to CSL360, a non Fc-engineered anti-CD123 mAb. The improved activity was evident as both an increased maximal level of target cell lysis and as a shift in the EC50 of the antibody to lower concentrations. Importantly, both primary AML blasts and CD34+CD38−CD123+LSC were susceptible to CSL362-induced ADCC and this was seen even in samples that were resistant to ADCC by a non Fc-engineered anti-CD123 mAb. In an AML xenograft mouse model, where treatment with the antibody was initiated 4 weeks after engraftment of leukemia cells, CSL362 was more effective in reducing leukemic growth than the non Fc-engineered anti-CD123 mAb. The evaluation of neutrophils, monocytes, macrophages and NK cells in ADCC assays has revealed that the major effector cell responsible for CSL362-mediated cytotoxicity in human peripheral blood is the NK cell. In clinical samples we have been able to demonstrate autologous depletion ex vivo of target AML blasts (collected at diagnosis and cryopreserved) following incubation with CSL362 and peripheral blood mononuclear cells (taken from the same patient at first remission), indicating that NK cell number and function is sufficiently preserved in such patients for CSL362-directed killing of leukemic target cells. The pre-clinical data generated thus far strongly support the clinical development of CSL362 for the treatment of AML in patients with adequate NK cell function. A Phase 1 study of CSL362 in patients with CD123 positive AML in remission is underway (Clinical Trials.gov identifier: NCT01632852). Disclosures: Busfield: CSL Limited: Employment. Biondo:CSL Limited: Employment. Wong:CSL Limited: Employment. Ramshaw:CSL Limited: Research Funding. Lee:CSL Limited: Research Funding. Martin:CSL Limited: Employment. Ghosh:CSL Limited: Employment. Braley:CSL Limited: Employment. Tomasetig:CSL Limited: Employment. Panousis:CSL Limited: Employment. Vairo:CSL Limited: Employment. Roberts:CSL Limited: Research Funding. DeWitte:CSL Behring: Employment. Lock:CSL Limited: Consultancy, Research Funding. Lopez:CSL Limited: Consultancy, Research Funding. Nash:CSL Limited: Employment.

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